Regulation of cell proliferation, differentiation, and migration is important for the formation and function of tissues. Regulatory proteins such as growth factors control these cellular processes and act as mediators in cell-cell signaling pathways. Growth factors are secreted proteins that bind to specific cell surface receptors on target cells. The bound receptors trigger intracellular signal transduction pathways which activate various downstream effectors that regulate gene expression, cell division, cell differentiation, cell motility, and other cellular processes. Some of the receptors involved in signal transduction by growth factors belong to the large superfamily of G-protein coupled receptors (GPCRs) which represent one of the largest receptor superfamilies known.
GPCRs are biologically important as their malfunction has been implicated in contributing to the onset of many diseases, which include, but are not limited to, Alzheimer's, Parkinson, diabetes, dwarfism, color blindness, retinal pigmentosa and asthma. Also, GPCRs have also been implicated in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure and in several cardiovascular, metabolic, neuro, oncology and immune disorders (F Horn, G Vriend, J. Mol. Med. 76: 464–468, 1998.). They have also been shown to play a role in HIV infection (Y Feng, C C Broder, P E Kennedy, E A Berger, Science 272:872–877, 1996).
GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which together form a bundle of antiparallel alpha (a) helices. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. These proteins range in size from under 400 to over 1000 amino acids (Strosberg, A. D. (1991) Eur. J. Biochem. 196: 110; Coughlin, S. R. (1994) Curr. Opin. Cell Biol. 6: 191–197). The amino-terminus of a GPCR is extracellular, is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops of GPCRs alternate with intracellular loops and link the transmembrane domains. Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account for structural and functional features of the receptor. In most G-protein coupled receptors, the bundle of a helices forms a ligand-binding pocket formed by several G-protein coupled receptor transmembrane domains.
The TM3 transmembrane domain has been implicated in signal transduction in a number of G-protein coupled receptors. Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled receptors, such as the b adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization. In fact, phosphorylation of an activated G-protein coupled receptor is a common mechanism for desensitizing signaling to a G-protein.
The extracellular N-terminal segment, or one or more of the three hydrophilic extracellular loops, have been postulated to face inward and form polar ligand binding sites which may participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor. In turn, the large, third intracellular loop of the activated receptor interacts with an intracellular heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or ion channel proteins. TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines have also been implicated in ligand binding (See, e.g., Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego Calif., pp. 2–6; Bolander, F. F. (1994) Molecular Endocrinology, Academic Press, San Diego Calif., pp. 162–176; Baldwin, J. M. (1994) Curr. Opin. Cell Biol. 6: 180–190; F Horn, R Bywater, G Krause, W Kuipers, L Oliveira, A C M Paiva, C Sander, G Vriend, Receptors and Channels, 5:305–314, 1998).
Recently, the function of many GPCRs has been shown to be enhanced upon dimerization and/or oligomerization of the activated receptor. In addition, sequestration of the activated GPCR appears to be altered upon the formation of multimeric complexes (AbdAlla, S., et al., Nature, 407:94–98 (2000)).
Structural biology has provided significant insight into the function of the various conserved residues found amongst numerous GPCRs. For example, the tripeptide Asp(Glu)-Arg-Tyr motif is important in maintaining the inactive confirmation of G-protein coupled receptors. The residues within this motif participate in the formation of several hydrogen bonds with surrounding amino acid residues that are important for maintaining the inactive state (Kim, J. M., et al., Proc. Natl. Acad. Sci. U.S.A., 94:14273–14278 (1997)). Another example relates to the conservation of two Leu (Leu76 and Leu79) residues found within helix II and two Leu residues (Leu 128 and Leu131) found within helix III of GPCRs. Mutation of the Leu128 results in a constitutively active receptor—emphasizing the importance of this residue in maintaining the ground state (Tao, Y. X., et al., Mol. Endocrinol., 14:1272–1282 (2000); and Lu. Z. L., and Hulme, E. C., J. Biol. Chem., 274:7309–7315 (1999). Additional information relative to the functional relevance of several conserved residues within GPCRs may be found by reference to Okada et al in Trends Biochem. Sci., 25:318–324 (2001).
GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, bombesin, bradykinin, endothelin, y-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., calcitonin, C5a anaphylatoxin, follicle stimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropin releasing hormone (TRH), and oxytocin). GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors.
GPCRs are implicated in inflammation and the immune response, and include the EGF module containing, mucin-like hormone receptor (Emrl) and CD97p receptor proteins. These receptors contain between three and seven potential calcium-binding EGF-like motifs (Baud, V. et al. (1995) Genomics 26: 334–344; Gray, J. X. et al. (1996) J. Immunol. 157: 5438–5447). These GPCRs are members of the recently characterized EGF-TM7 receptors family. In addition, post-translational modification of aspartic acid or asparagine to form erythro-p-hydroxyaspartic acid or erythro-p-hydroxyasparagine has been identified in a number of proteins with domains homologous to EGF. The consensus pattern is located in the N-terminus of the EGF-like domain. Examples of such proteins are blood coagulation factors VII, IX, and X; proteins C, S, and Z; the LDL receptor; and thrombomodulin.
One large subfamily of GPCRs are the olfactory receptors. These receptors share the seven hydrophobic transmembrane domains of other GPCRs and function by registering G protein-mediated transduction of odorant signals. Numerous distinct olfactory receptors are required to distinguish different odors. Each olfactory sensory neuron expresses only one type of olfactory receptor, and distinct spatial zones of neurons expressing distinct receptors are found in nasal pasages. One olfactory receptor, the RAlc receptor which was isolated from a rat brain library, has been shown to be limited in expression to very distinct regions of the brain and a defined zone of the olfactory epithelium (Raming, K. et al., (1998) Receptors Channels 6: 141–151). In another example, three rat genes encoding olfactory-like receptors having typical GPCR characteristics showed expression patterns exclusively in taste, olfactory, and male reproductive tissue (Thomas, M. B. et al. (1996) Gene 178: 1–5).
Another group of GPCRs are the mas oncogene-related proteins. Like the mas oncogenes themselves, some of these mas-like receptors are implicated in intracellular angiotensin II actions.
Angiotensin II, an octapeptide hormone, mediates vasoconstriction and aldosterone secretion through angiotensin II receptor molecules found on smooth vascular muscle and the adrenal glands, respectively.
A cloned human mas-related gene (mrg) mRNA, when injected into Xenopus oocytes, produces an increase in the response to angiotensin peptides. Mrg has been shown to directly affect signaling pathways associated with the angiotensin II receptor, and, accordingly, affects the processes of vasoconstriction and aldosterone secretion (Monnot, C. et al. (1991) Mol. Endocrinol. 5: 1477–1487).
GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Rhodopsin is the retinal photoreceptor which is located within the discs of the eye rod cell. Parma, J. et al. (1993, Nature 365: 649–651) reported that somatic activating mutations in the thyrotropin receptor cause hyperfunctioning thyroid adenomas and suggested that certain GPCRs susceptible to constitutive activation may behave as protooncogenes.
Purines, and especially adenosine and adenine nucleotides, have a broad range of pharmacological effects mediated through cell-surface receptors. For a general review, see Adenosine and Adenine Nucleotides in The G-Protein Linked Receptor Facts Book, Watson et al. (Eds.) Academic Press 1994, pp. 19–31.
Some effects of ATP include the regulation of smooth muscle activity, stimulation of the relaxation of intestinal smooth muscle and bladder contraction, stimulation of platelet activation by ADP when released from vascular endothelium, and excitatory effects in the central nervous system. Some effects of adenosine include vasodilation, bronchoconstriction, immunosuppression, inhibition of platelet aggregation, cardiac depression, stimulation of nociceptive afferants, inhibition of neurotransmitter release, pre- and postsynaptic depressant action, reducing motor activity, depressing respiration, inducing sleep, relieving anxiety, and inhibition of release of factors, such as hormones.
Distinct receptors exist for adenosine and adenine nucleotides. Clinical actions of such analogs as methylxanthines, for example, theophylline and caffeine, are thought to achieve their effects by antagonizing adenosine receptors. Adenosine has a low affinity for adenine nucleotide receptors, while adenine nucleotides have a low affinity for adenosine receptors.
There are four accepted subtypes of adenosine receptors, designated A1, A2A, A2B, and A3. In addition, an A4 receptor has been proposed based on labeling by 2 phenylaminoadenosine (Cornfield et al. (1992) Mol. Pharmacol. 42: 552–561).
P2x receptors are ATP-gated cation channels (See Neuropharmacology 36 (1977)). The proposed topology for PZX receptors is two transmembrane regions, a large extracellular loop, and intracellular N and C-termini.
Numerous cloned receptors designated P2y have been proposed to be members of the G-protein coupled family. UDP, UTP, ADP, and ATP have been identified as agonists. To date, P2Y1–7 have been characterized although it has been proposed that P2Y7 may be a leukotriene B4 receptor (Yokomizo et al. (1997) Nature 387: 620–624).
It is widely accepted, however, that P2Y 1, 2, 4, and 6 are members of the G-protein coupled family of P2y receptors.
At least three P2 purinoceptors from the hematopoietic cell line HEL have been identified by intracellular calcium mobilization and by photoaffinity labeling (Akbar et al. (1996) J. Biochem. 271: 18363–18567).
The Ai adenosine receptor was designated in view of its ability to inhibit adenylcyclase. The receptors are distributed in many peripheral tissues such as heart, adipose, kidney, stomach and pancreas. They are also found in peripheral nerves, for example intestine and vas deferens. They are present in high levels in the central nervous system, including cerebral cortex, hippocampus, cerebellum, thalamus, and striatum, as well as in several cell lines. Agonists and antagonists can be found on page 22 of The G-Protein Linked Receptor Facts Book cited above, herein incorporated by reference. These receptors are reported to inhibit adenylcyclase and voltage-dependent calcium chanels and to activate potassium chanels through a pertussis-toxin-sensitive G-protein suggested to be of the G/Go class. Ai receptors have also been reported to induce activation of phospholipase C and to potentiate the ability of other receptors to activate this pathway.
The A2A adenosine receptor has been found in brain, such as striatum, olfactory tubercle and nucleus accumbens. In the periphery, A2 receptors mediate vasodilation, immunosuppression, inhibition of platelet aggregation, and gluconeogenesis. Agonists and antagonists are found in The G-Protein Linked Receptor Facts Book cited above on page 25, herein incorporated by reference. This receptor mediates activation of adenylcyclase through Gs.
The A2B receptor has been shown to be present in human brain and in rat intestine and urinary bladder. Agonists and antagonists are discussed on page 27 of The G-Protein Linked Receptor Facts Book cited above, herein incorporated by reference. This receptor mediates the stimulation of cAMP through Gg.
The A3 adenosine receptor is expressed in testes, lung, kidney, heart, central nervous system, including cerebral cortex, striatum, and olfactory bulb. A discussion of agonists and antagonists can be found on page 28 of The G-Protein Linked Receptor Facts Book cited above, herein incorporated by reference. The receptor mediates the inhibition of adenylcyclase through a pertussis-toxin-sensitive G-protein, suggested to be of the Gi/Go class.
The P2Y purinoceptor shows a similar affinity for ATP and ADP with a lower affinity for AMP. The receptor has been found in smooth muscle, for example, and in vascular tissue where it induces vasodilation through endothelium-dependent release of nitric oxide. It has also been shown in avian erythrocytes.
Agonists and antagonists are discussed on page 30 of The G-Protein Linked Receptor Facts Book cited above, herein incorporated by reference. The receptor function through activation of phosphoinositide metabolism through a pertussis-toxin insensitive G-protein, suggested to be of the Gi/Go class.
N-formyl peptide receptors are another family of G-protein coupled receptors. N-formyl peptide receptors are known to bind to N-formyl peptides and mediate a number of host defensive responses of human neutrophils that result in chemotaxis, secretion of hydrolytic enzymes, and superoxide generation. Inappropriate activation or defective regulation of these responses can result in pathogenic states responsible for inflammatory disease. The receptor is a 50 to 70-kD, integral plasma membrane glycoprotein with intracellular and surface localization. Its abundance in the membrane is regulated by membrane flow and recycling processes. Cytoskeletal interactions are believed to control its organization in the plane of the membrane and interaction with other proteins. The receptor's most important interaction is with guanyl nucleotide binding proteins that serve as signal transduction partners ultimately leading to activation of effector responses. Because the interaction of the receptor with G proteins is necessary for transduction, control of this interaction may be at the root of understanding the molecular control of responses in these cells (Jesaitis, A, J., Allen, R, A, J. Bioenerg, Biomembr., 20(6):679–707, (1988)).
Two particular proteins, Formyl Peptide Receptor (FPR) and Formyl Peptide Receptor-Like 1 (FPRL1) belong to the seven-transmembrane, GPCR superfamily. Because of the capacity of FPR and FPRL1 to interact with bacterial chemotactic formylated peptides, these receptors are thought to play a role in host defense against microbial infection. In addition, a variety of novel agonists have been identified for these receptors, including several endogenous molecules that are involved in proinflammatory responses. Most notably, FPRL1 has been found to be used by at least three amyloidogenic protein and peptide ligands, the serum amyloid A (SAA), the 42 amino acid form of □ amyloid (A beta42), and the prion peptide PrP106–126, to chemoattract and activate human phagocytic leukocytes (Su et al., J. Exp. Med., 189:395–402 (1999), Le et al. J. Neurosci., 21(RC123):1–5 (2000), Le et al., J. Immunol., 166:1448–1451 (2001), Chiang et al., J. Exp. Med., 191:1197–1207 (2000), Yang et al., J. Exp. Med. 192:1069–1074 (2000)). These findings have greatly expanded the functional scope and role in pathophysiological conditions for the formyl peptide receptors.
Leukocyte or phagocyte recruitment at sites of infection and inflammation is dependent on the presence of a gradient of chemotactic factors or chemoattractants. Chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (fMLF), activated complement component 5 (C5a), leukotriene B4 (LTB4), platelet-activating factor (PAF) and chemokines activate GPCRs on both hematopoietic and non-hematopoietic cells (Hwang, J. Lipid Med., 2:123–158 (1990); Oppenheim et al., Annu. Rev. Immunol., 9:617–648 (1991), Rollins, Blood, 90:909–26 (1997), Zlotnik et al., Crit. Rev. Immunol., 19:1–47 (1999)). Synthetic fMLF was one of the first identified leukocyte chemoattractants. Natural fMLF was subsequently purified and identified in supernatants of gram negative bacteria. fMLF activates FPR on leukocytes to increase cell migration, phagocytosis and the release of proinflammatory mediators. Since picomolar to nanomolar amounts of fMLF can activate FPR, and high concentrations of fMLF can only elicit a limited response upon FPRL1, FPR is considered to be a high-affinity receptor, while FPRL1 is considered to be a low-affinity receptor (Ye et al., Biochem. Biophys. Res. Commun., 184:582–589 (1992), Durstin et al, Biochem. Biophys. Res. Commun., 201:174–179 (1994)).
FPRL1 possesses 69% identity at the amino acid level with FPR (Boulay et al., Biochem. Biophys. Res. Commun., 168:1103–1109 (1990), Boulay et al., Biochemistry, 29:11123–11133 (1990)). FPR is expressed on phagocytic leukocytes, and FPRL1 is expressed on phagocytic leukocytes and hepatocytes, epithelial cells, T lymphocytes, astrocytoma cells, neuroblastoma cells, and microvascular endothelial cells (Prossnitz et al., Pharmacol. Ther., 74:73–102 (1997), Le et al., J. Neuroimmunol., 111:102–108 (2000), Ye et al., Biochem. Biophys. Res. Commun., 184:582–589 (1992), Gronert et al., J. Exp. Med., 187:1285–1294 (1998)).
A variety of ligands have been identified for formyl peptide receptors. In addition to bacterial fFML, a number of synthetic peptides activate FPR and FPRL1, such as the hexapeptide WKYMVM (SEQ ID NO:99) (Bae et al., J. Leuko. Bio, 65:241–248 (1999)). Also, HIV envelope proteins contain domains that interact with formyl peptide receptors (Su et al, J. Immunol., 162:5924–5930 (1999)). Lastly, a great variety of endogenous ligands interact with FPRL1, such as serum amyloid A (SAA), the 42 amino acid form of beta amyloid (A beta42), prion peptide PrP106–126, mitochondria peptide fragment MYFINILTL (SEQ ID NO:100) from NADH dehydrogenase subunit 1, and LL-37, an enzymatic cleavage fragment of the neutrophil granule-derived cathelicidin (Su et al., J. Exp. Med., 189:395–402 (1999), Le et al. J. Neurosci., 21(RC123):1–5 (2000), Le et al., J. Immunol., 166:1448–1451 (2001), Chiang et al., J. Exp. Med., 191:1197–1207 (2000), Yang et al., J. Exp. Med, 192:1069–1074 (2000)). All of these ligands are chemotactic and illicit proinflammatory responses in human leukocytes. LL-37 is also an antimicrobial peptide.
SAA, A beta42 and PrP106–126 are endogenous proteins that tend to precipitate when aggregated and result in amyloid deposition in pathologic states such as systemic amyloidosis (SAA), Alzheimer's disease (A beta42) and prion disease (PrP106–126). Thus the recent identification of novel and host-derived ligands for both FPR and FPRL1 suggests that these receptors may contribute to the proinflammatory aspects of systemic amyloidosis and neurodegenerative diseases.
Using the above examples, it is clear the availability of a novel cloned G-protein coupled receptor provides an opportunity for adjunct or replacement therapy, and are useful for the identification of G-protein coupled receptor agonists, or stimulators (which might stimulate and/or bias GPCR action), as well as, in the identification of G-protein coupled receptor inhibitors. All of which might be therapeutically useful under different circumstances.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of HGPRBMY39 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the HGPRBMY39 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.